Journal of Microscopy, Vol. 227, Pt 3 September 2007, pp. 203–215
Received 14 March 2007; accepted 30 May 2007
A white light confocal microscope for spectrally resolved
multidimensional imaging
J. H . F R A N K ∗ , A . D. E L D E R †, J. S WA RT L I N G †,
A . R . V E N K I TA R A M A N ‡, A . D. J E YA S E K H A R A N ‡
& C . F. K A M I N S K I †
∗ Combustion Research Facility, Sandia National Laboratories, Livermore, CA 94611, USA
†Department of Chemical Engineering, University of Cambridge, Cambridge, UK
‡MRC Cancer Cell Unit, Hutchinson/MRC Research Centre, Cambridge, UK
Key words. Confocal microscopy, fluorescence microscopy, photonic crystal
fibre, supercontinuum.
Summary
Spectrofluorometric imaging microscopy is demonstrated in
a confocal microscope using a supercontinuum laser as an
excitation source and a custom-built prism spectrometer for
detection. This microscope system provides confocal imaging
with spectrally resolved fluorescence excitation and detection
from 450 to 700 nm. The supercontinuum laser provides
a broad spectrum light source and is coupled with an
acousto-optic tunable filter to provide continuously tunable
fluorescence excitation with a 1-nm bandwidth. Eight different
excitation wavelengths can be simultaneously selected. The
prism spectrometer provides spectrally resolved detection with
sensitivity comparable to a standard confocal system. This
new microscope system enables optimal access to a multitude
of fluorophores and provides fluorescence excitation and
emission spectra for each location in a 3D confocal image. The
speed of the spectral scans is suitable for spectrofluorometric
imaging of live cells. Effects of chromatic aberration are modest
and do not significantly limit the spatial resolution of the
confocal measurements.
Introduction
Confocal fluorescence microscopy is one of the most powerful
techniques for probing a wide range of phenomena in biological
sciences (Pawley, 1995). The photophysical properties of a
fluorophore are affected by interactions of the fluorophore
with its local environment, resulting in variations of
fluorescence intensity, lifetime, anisotropy, and emission and
excitation spectra. These interactions represent opportunities
for sensitive probing of local environmental properties,
Correspondence to: Jonathan H. Frank. Tel: +1 925 294 4645; fax: +1 925 294
2595; e-mail: [email protected]; [email protected]
C 2007 The Royal Microscopical Society
Journal compilation No claim to original US government works
such as pH, viscosity, and the presence of quenchers, and
enable detection of phenomena, such as binding events or
conformational changes in macromolecules.
The applications of fluorescence microscopy are rapidly
expanding as scientists probe increasingly complex and
varied biological systems. These applications require
advances in microscopy technology. Developments of
new fluorophores are significantly enhancing labelling
specificity and providing new opportunities for studying
complex phenomena using simultaneous measurements of
multiple different fluorophores. Advancements in microscope
instrumentation are required to implement these multiplexed
fluorophore measurements and to provide a more complete
characterization of the interactions of fluorophores with their
local environment.
The demand for more sophisticated and versatile microscopy
systems requires innovative coupling of microscopes with
advanced laser and detector technologies. These technologies
can provide unprecedented detection sensitivity and flexibility
for fluorescence measurements. Until recently, the inability
to arbitrarily choose fluorescence excitation, and detection
wavelengths and bandwidths presented a bottleneck in
the advancement of confocal laser scanning microscopy
(CLSM). Currently, most commercial confocal systems use a
combination of lasers to excite fluorescence at a small number
of discrete wavelengths in the visible spectrum. However,
flexibility in wavelength selection is essential for optimal
excitation and detection of fluorophores (Zimmermann et al.,
2003).
Full flexibility in targeting any wavelength interval in
the visible spectrum enables simultaneous measurements
of different species, structures and phenomena using
multiplexed detection of several fluorophores (Stephens
et al., 2000). The ability to rapidly change between any
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J. H . F R A N K E T A L .
visible excitation wavelength greatly enhances the use
of photo-activated dyes (Lippincott-Schwartz et al., 2003;
Betzig et al., 2006). Spectrally resolved fluorescence
measurements are essential for differentiating multiple
fluorophore signals from one another and from interferences,
such as autofluorescence. The spectral differentiation of
autofluorescence signals enables entirely new diagnostic
opportunities. For example, measurements at multiple
excitation and emission wavelengths may significantly
enhance the use of autofluorescence as a diagnostic tool for
applications such as in vivo differentiation of healthy and
diseased tissue. This approach would provide image contrast
without requirements for labelling (Zellweger et al., 2001). A
similar image contrasting capability has been demonstrated
with fluorescence lifetime imaging (Tadrous et al., 2003).
Currently, the most commonly used excitation sources for
CLSM systems are gas lasers, such as Ar-ion or He-Ne lasers,
and diode lasers for operation in the visible to near-UV spectral
regions. For emission detection, photomultiplier tubes (PMT)
are normally used in conjunction with dichroic mirrors and
bandpass filters for spectral selection. These configurations
create spectrally inflexible systems, and only recently have
solutions emerged to address these problems.
For fluorescence excitation, novel laser sources that generate
white light supercontinua show excellent potential for
CLSM imaging applications. For microscopy applications,
supercontinua are usually generated by pumping a photonic
crystal fibre (PCF; Dudley et al., 2006) or a tapered silica fibre
with ultrashort pulses from a Ti:sapphire laser (Dunsby et al.,
2004; McConnell, 2004; Betz et al., 2005; Palero et al., 2005).
Once a supercontinuum is generated, the desired excitation
wavelength must be selected. Techniques for wavelength
selection include bandpass filters (McConnell, 2004), spatial
dispersion of the supercontinuum with a prism (Dunsby
et al., 2004), acousto-optic beam splitters (AOBS; Betz et al.,
2005) and acousto-optic tunable filters (AOTF; Borlinghaus
et al., 2006). The use of bandpass filters is straightforward
to implement but inflexible. Spatial dispersion of the beam
using a prism can lead to large losses and does not allow
simultaneous excitation with multiple excitation lines. The
AOTF is a well-established technology that provides computercontrolled multiline wavelength selection over a broad spectral
range.
For emission detection, a grating (Schultz et al., 2001) or
prism (Frederix et al., 2001) is used to spatially disperse the
different emission wavelengths, and the dispersed spectrum
is imaged onto an array detector (Sinclair et al., 2006). The
array detector provides rapid acquisition of an entire emission
spectrum but may limit the detection sensitivity in low-signal
applications. An alternative approach is to pass the spatially
dispersed emission through an adjustable slit system and onto
a PMT. The emission spectra are measured sequentially by
scanning the slit position. Although this approach is slower
than the array detector, it provides flexibility for improving
the detection limit in low-signal applications by increasing
the bandwidth of the detection (Borlinghaus et al., 2006).
For specialized CLSM applications, simultaneous time- and
wavelength-resolved detection is possible with time- and
position-sensitive detector systems (Plessow et al., 2000; Luong
et al., 2005).
Here we describe the design and first full calibration of
a confocal microscope system with continuously tunable
fluorescence excitation and emission wavelengths and
bandwidths. This system has the flexibility for optimal
excitation and detection of a wide range of fluorophores and
provides a new capability for multiparameter fluorescence
measurements. Previous work has shown the potential
of multiparameter fluorescence as an analytical tool
(Borlinghaus et al., 2006). The excitation and detection
systems are fully integrated into a commercial confocal
microscope while retaining all of its intrinsic features,
such as three-dimensional (3D) imaging, frame and line
averaging, and region-of-interest scanning. For an excitation
source, we use a compact fibre-laser pumped PCF that
generates supercontinuum radiation. This laser technology
is a promising alternative to the more complex and
expensive Ti:sapphire-based supercontinuum systems. For
spectrally resolved detection, we use a custom-designed prism
spectrometer with a computer-controlled slit mechanism. The
spectral and spatial resolutions of the system are assessed. The
integrated microscope system is optimized for light throughput
and has sensitivity and spatial resolution that is comparable to
top-of-the-range commercial CLSM systems. We demonstrate
high-resolution spectrofluorometric measurements with voxel
sizes of less than 0.1 femtoliters.
Experimental methods
The spectrofluorometric imaging system is constructed
around a commercial confocal microscope scanning unit
(Olympus FluoView 300) coupled with an Olympus IX70
inverted microscope frame (Olympus UK, Southall, UK). All
lasers are removed from the original CLSM frame, and the unit
is modified to accept an external light source for fluorescence
excitation. A fibre optic signal collection system has been
designed to transmit signals from the confocal pinhole to
the spectrograph for detection. A schematic diagram of
the integrated system is shown in Fig. 1. The excitation,
detection and image acquisition are fully computer controlled.
The confocal scanning and image acquisition are controlled
with Olympus’ proprietary software (FluoView ver. 4.3 with
TIEMPO) (Olympus UK, Southall, UK) on one computer.
A second computer controls the fluorescence excitation
and detection wavelengths with a custom-designed LabView
(National Instruments UK, Newbury, UK) application. For performing fluorescence excitation and emission scans, the excitation, detection and confocal subsystems are synchronized by
sending triggering signals between the two computers.
C 2007 The Royal Microscopical Society
Journal compilation No claim to original US government works, Journal of Microscopy, 227, 203–215
W H I T E L I G H T C O N F O C A L M I C RO S C O P E
Excitation
Detection
Photodiode
Fiber laser
Photonic crystal
fiber
205
Acousto-optic
tunable filter
Prism
spectrometer
Confocal
microscope
Fluorescence
Emission
Slit
Detector
Computer
Fig. 1. Experimental configuration of multispectral confocal imaging microscope.
The output from a supercontinuum laser (Fianium
SC450, Fianium, Southampton, UK) is passed through
an AOTF to provide continuously tunable fluorescence
excitation throughout the visible spectrum. The full
spectrum of the supercontinuum laser spans 450–
1700 nm with a total output power of 4 W. In the present
configuration, the infrared radiation is removed from the
beam by dichroic mirrors, and only the visible portion of the
spectrum is used. The supercontinuum laser system produces
approximately 5-ps pulses at 40 MHz and consists of a master
laser, an amplifier and a PCF. The low-power master laser is
a passively mode locked Yb-doped core-pumped fibre laser
(λ = 1060 nm). The master laser is coupled to a high-power
cladding-pumped fibre amplifier. The amplified output pumps
a high nonlinearity PCF, which generates a broad spectrum of
spatially coherent light by a combination of nonlinear optical
effects and anomalous group velocity dispersion (Dudley
et al., 2006). Nonlinear effects, such as self-modulation,
cross-modulation, four-wave mixing and stimulated Raman
scattering, are induced by intense laser light that is confined to
the small-diameter core of the microstructured fibre. Figure 2a
shows the visible region of the PCF-generated spectrum with
different pump powers. The pump power is varied by adjusting
the fibre amplifier gain. At low powers, only the red portion of
the spectrum is present. As the power is increased, the shorter
wavelength regions of the spectrum fill in. At maximum
power, a prominent peak appears at approximately 480 nm,
and a minor isolated peak emerges at 432 nm.
The AOTF filter provides a continuously adjustable bandpass
filter that selects lines from the supercontinuum spectrum.
As many as eight bands can be selected simultaneously with
each band having approximately a 1-nm bandwidth. The
intensity and the centre wavelength of each band can be
independently controlled by changing the acoustic power
and modulation frequency, respectively. The bandpass can be
tuned from 450 to 700 nm by varying the AOTF modulation
frequency from 80 to 150 MHz. The AOTF driver is computer
controlled via an RS-232 serial connection using LabView.
Figure 2b shows a power spectrum that was measured by
tuning a single transmission band of the AOTF from 450 to
650 nm. This spectrum was recorded with the fibre amplifier
at maximum power. If additional power is required, multiple
closely spaced bandpasses can provide significantly greater
than 1 mW total power at any visible wavelength. Figure 3
illustrates the multiline capabilities of the AOTF with a
spectrum that was produced by programming the AOTF to
transmit two separate combs in the blue and green regions
with four lines in each comb. The width of the individual
transmission lines was less than or comparable to the 1–2
nm resolution of the spectrometer that was used to measure
the spectrum (Ocean Optics USB2000, Ocean Optics, Dunedin,
FL, USA). The minimum wavelength separation between two
Fig. 2. (a) Visible portion of the supercontinuum spectrum generated by the photonic crystal fibre with different pumping powers. Black curve corresponds
to the maximum laser power. (b) AOTF-transmitted power for a single line with full laser power.
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J. H . F R A N K E T A L .
Fig. 3. Example of a multiline excitation spectrum using a programmable
AOTF to simultaneously select eight different lines.
Fig. 4. Transmission spectrum from two different AOTF drivers with and
without sideband transmission.
lines is limited by the minimum spacing of the corresponding
acoustic modulation frequencies. The minimum separation
of the modulation frequencies for two adjacent lines is
approximately 500 kHz, which corresponds to 1–4 nm
depending on the wavelength. For smaller line separations, the
beating between the acoustic frequencies produces significant
intensity fluctuations in the transmitted light, which can
create stripes in the confocal images.
The multiline capability may provide sufficient peak
intensities for two-photon fluorescence microscopy in some
applications. The use of all eight AOTF lines provides
approximately 8 mW of 5-ps pulses at 40 MHz in the red region,
and this capability could be extended to the near infrared with
a different AOTF. Many commercial two-photon microscopy
systems use Ti:sapphire lasers with subpicosecond pulse
durations and higher repetition rates (Denk et al., 1995). Palero
et al. (2005) demonstrated that two-photon fluorescence
excitation is possible with supercontinua generated from a
Ti:sapphire-pumped PCF. To compare our supercontinuum
system with commercial Ti:sapphire systems, we consider
the parameters that govern the two-photon laser-induced
fluorescence (LIF) signal. For a fixed pixel dwell time, the twophoton LIF signal is proportional to P 2peak f τ , or equivalently
to P̄ 2 / f τ , where Ppeak and P̄ are the peak and average
laser powers, respectively, f is the pulse repetition rate and
τ is the pulse width. Using this relationship, we estimate
that two-photon LIF excitation with P̄ = 8 mW from the
supercontinuum laser would produce a LIF signal that is
comparable to excitation with P̄ = 2.5 mW from a Ti:sapphire
laser with 250-fs pulses and a repetition rate of 80 MHz. For
some applications, this power is sufficient for two-photon LIF
imaging, whereas other applications require larger powers
(Konig, 2000; Diaspro et al., 2001). Note that the actual
two-photon excitation efficiency will depend on a number
of parameters, including the two-photon cross-section of the
fluorophore, the temporal coherence of different wavelengths
within the supercontinuum laser spectrum as well as the
overlap of these wavelengths with the fluorophore’s absorption
spectrum.
The spectral purity of the transmitted light is another
issue that must be considered when using an AOTF to filter
a broadband light source. The narrow-band transmission
spectrum of a single line can be degraded by the presence of
sidebands that arise from the distribution of the acoustic field in
the acousto-optic crystal. These sidebands can be particularly
problematic if the output of the acousto-optic driver electronics
contains excess frequency components. Sidebands can be
significantly attenuated by using an additional AOTF, or a
second pass through the same AOTF. Figure 4 shows the
transmission spectrum for a bandpass centered at 465 nm
using two different AOTF drivers. One of the AOTF drivers
produced a significant sideband that was offset from the main
band by approximately 30 nm. This sideband was eliminated
by a double-pass configuration that was implemented by
retroreflecting the beam through the AOTF. The disadvantage
of using a second pass is that the transmission of the primary
line is reduced by approximately 50%. The second AOTF driver
provided an improved acoustic spectrum and eliminated this
sideband. In this case, a single pass through the AOTF was used
with negligible contributions from sidebands.
The output beam from the AOTF is aligned onto the
confocal scanning mirrors after being reflected by a 20/80
(R/T) broadband beamsplitter. In the present setup, the 20/80
beamsplitter replaces the excitation dichroic beamsplitter
that is used in the standard configuration of the confocal
microscope. The total throughput of the optical path for
the excitation beam, including the objective lens, mirrors
and the 20/80 beamsplitter, varied from 8–10% over the
visible spectrum. The 20/80 beamsplitter was chosen to
pass the maximum signal over the entire visible spectrum
while minimizing nonlinear effects, such as photobleaching
or saturation of the fluorophores. If nonlinear effects are
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negligible and the fluorescence detection sensitivity is limited
by the available laser intensity, the signal levels in the CLSM
can be increased by approximately 56% by replacing the 20/80
beamsplitter with a 50/50 beamsplitter.
Fluorescence emission is detected using a high-efficiency
prism spectrometer that is based on the design by Frederix
et al. (Frederix et al., 2001). The spectrometer is coupled to the
confocal microscope using a multimode optical fibre (105 µm
core diameter, NA = 0.22). The microscope objective images
the fluorescence from the sample onto the standard confocal
aperture. The confocal aperture is imaged onto the input of
the optical fibre using an antireflection (AR)-coated doublet
achromat (f.l. = 20 mm) to minimize chromatic aberrations.
Ray tracing software (Winlens 4.3, Linos Photonics, Milton
Keynes, UK) was used to optimize the imaging of the pinhole
onto the fibre by matching the numerical apertures of the
imaging lens and the fibre. The output of the optical fibre is
positioned at the focal point of the spectrometer objective lens
(achromat, f.l. = 120 mm). The collimated light that emerges
from the objective lens is dispersed by a 60-mm AR-coated
equilateral SF10 prism. The dispersed spectrum is focused onto
the spectrometer exit slit by the spectrometer imaging lens
(achromat, f.l. = 80 mm). A sidewindow PMT (Hamamatsu
R3896, Hamamatsu Photonics UK, Welwyn Garden City, UK)
is positioned at the spectrometer exit slit. The imaging system
is designed to yield a maximum spatial dispersion of 80 mm in
the slit plane (see Fig. 5) to prevent overfilling of the active area
of the detector. The exit slit system is custom built and consists
of two knife edges mounted in a linear stage. The wavelength
and bandwidth of the emission detection are controlled by
positioning the knife edges using computer-controlled stepper
motors. The slit width can be adjusted from 25 µm to fully
open (8 mm) with 25-µm resolution using a digital counter
card and control software from National Instruments.
The prism spectrometer design calculations are compared
with measurements of the spectrometer’s dispersion. For a
given spectral bandpass, δλ, the image size x in the slit plane
varies as
d θD d n
δλ,
(1)
x = f
dn dλ
where f is the focal length of the spectrometer imaging lens
(80 mm), θ D is the deviation angle and n the refractive index
(Born & Wolf, 1980). The dispersion dn/dλ is calculated using
Sellmeier’s dispersion formula:
Fig. 5. Comparison of measured and calculated dispersion curves for prism
spectrometer.
Fig. 6. Calculated spectrometer resolution for entrance slit widths of 50,
100 and 300 µm.
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B1 λ2
B2 λ2
B3 λ2
+
+
,
(2)
λ2 − C 1
λ2 − C 2
λ2 − C 3
where B i and C i are dispersion coefficients for SF10 (SCHOTT,
2006). The spectrometer is operated at a fixed input angle
under minimum deviation (Klein & Furtak, 1986) at 543 nm,
which corresponds to the wavelength of the helium neon laser
that is used to align the spectrometer.
Figure 5 shows the excellent agreement between the
computed and measured dispersion curves. The centre
wavelength for each position of the slit is measured by
illuminating a blank slide with a single line from the AOTF
and detecting elastically scattered light while the AOTF
wavelength is scanned. The theoretical spectral resolution
of the spectrometer can be determined from the derivative
of the dispersion curve. The dispersion of SF10 is larger at
shorter wavelengths, and consequently the spectral resolution
increases as the wavelength decreases. For example, a spectral
bandpass of δλ = 1 nm corresponds to slit sizes that vary
approximately from x = 65 µm to 8 µm at λ = 450 and
λ = 750 nm, respectively. In practice, the spectral resolution is
limited by the slit width and the diameter of the optical fibre at
the input of the spectrometer. Figure 6 shows a calculation of
the spectral resolution for fibre diameters from 50 to 300 µm.
The selection of the fibre diameter is a compromise between
spectral resolution and detection sensitivity. The use of larger
fibres improves the detection sensitivity by enabling more
efficient coupling from the confocal aperture but decreases the
n 2 (λ) − 1 =
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J. H . F R A N K E T A L .
spectral resolution. For the confocal images presented here,
we use a 105 µm fibre, which provides spectral resolutions
of 1.5 nm for detection at 450 nm and 6 nm at 700 nm. The
overall transmission of the spectrometer, including the 105 µm
fibre, was approximately 75% across the visible spectrum.
The sensitivity of the spectrometer is thus better than or
comparable to the standard confocal detector configuration,
which requires the use of dichroic and bandpass filters to
block background scattering. These filters can significantly
reduce the detected signal, and a large set of filter combinations
is required to optimize the detection sensitivity for different
fluorophores.
(a)
Results and discussion
Characterization of fluorophores with fluorescence emission and
excitation scans
In situ confocal imaging of fluorescence excitation and
emission spectra are an important tool for characterizing
fluorophores and using multiple fluorophores to label a
sample. As a demonstration of this capability, we performed
fluorescence excitation and emission scans of three dyes
with the confocal microscope system. Figure 7 shows
measured excitation and emission spectra of Rhodamine
6G, Rhodamine B and Coumarin 6 dyes, whose combined
fluorescence spectra cover much of the visible spectrum.
For these measurements, solutions of the dyes in ethanol
were placed in multiwell plates on the microscope stage. The
spectra measured with the confocal system are compared
with absorption and emission spectra that were measured
with a commercial spectrometer. The excellent agreement
between these reference spectra and the spectra measured
with the confocal system is evident in Fig. 7. These
results demonstrate that precision spectrofluorometry can be
performed at diffraction-limited resolution and that even subtle
changes in absorption/emission features can be differentiated
with this instrument. An exciting opportunity exists for
identifying molecules from such spectral signatures without
any requirement for labelling the sample.
Accurate spectral scans require careful corrections for
variations in the excitation laser power, detection bandwidth
and detector sensitivity. The scans presented here are corrected
for these factors with the corrected fluorescence signal given
by
Scorr = Smeas
C
, (3)
Plaser Texc (λexc )Rdet (λdet )Tdet (λdet )λdet
where S meas is the measured fluorescence signal, C is a
calibration constant, P laser is the laser power, λ exc and λ det are
the excitation and detection wavelengths, respectively, Texc (λ)
is the total transmittance of the optical train that directs the
laser beam to the sample, Rdet (λ) is the detector sensitivity,
Tdet (λ) is the total transmittance of the detection optical train
(b)
(c)
Fig. 7. Excitation and emission spectra of (a) Rhodamine 6G, (b)
Rhodamine B, (c) Coumarin 6 measured with the multispectral confocal
microscope. Results are compared with reference absorption and emission
spectra measured using a commercial spectrometer.
and λ det is the detection bandwidth of the spectrometer.
Equation (3) is valid for narrow detection bandwidths for
which the product Rdet (λ)Tdet (λ) is approximately constant.
These corrections are particularly important for scans that
cover a large range of wavelengths. As an illustration of the
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W H I T E L I G H T C O N F O C A L M I C RO S C O P E
(a)
209
(b)
Fig. 8. (a) Confocal image of a mixture of microbeads containing three different fluorescent dyes. The different beads are identified by a fluorescence
excitation scan and are displayed with corresponding blue, green and red colour palettes. (b) Fluorescence excitation spectra are depicted for the blue,
green and red beads.
quality of the corrections in these dye spectra, we consider
the Coumarin 6 excitation spectrum, which spans a range
of the supercontinuum spectrum that includes a significant
peak at 480 nm (see Fig. 2). The dashed line in Fig. 7c shows
an excitation spectrum that was measured with the confocal
system prior to the laser power correction. Despite the large
variation in the excitation power, the corrected spectrum
(solid line) shows excellent agreement with the reference
spectrum. For the excitation scans presented here, the emission
spectra remained relatively constant, allowing for broadband
detection without corrections for variations in Rdet (λ)Tdet (λ).
However, corrections for variations in Rdet (λ)Tdet (λ) and the
spectrometer detection bandwidth are required for scans of the
emission spectra. In principle, it is possible to keep the detection
bandwidth of the instrument constant during a spectral scan
by adjusting the separation of the knife edges. For the scans
shown in this paper, however, both stepper motors are moved
at the same speed resulting in a constant slit width and a
variable bandpass (see Fig. 6) for which the measured spectra
are corrected using Eq. 3. The excellent agreement between the
reference and corrected emission spectra in Fig. 7 illustrates
this correction capability.
Multiplexed fluorophore measurements
The multiplexing capability of the confocal microscope system
is illustrated by imaging a mixture of microbeads containing
three different fluorescent dyes. The beads are 0.18-µm
diameter PS-Speck beads (Molecular Probes, Invitrogen,
Carlsbad, CA, USA) with excitation and emission maxima
at approximately 505/515 nm, 540/560 nm and 633/660
nm. A fluorescence excitation scan is performed using
two-dimensional (2D) confocal imaging. At each excitation
wavelength, a 29 µm × 29 µm region is imaged with a
60× objective lens (Olympus PlanApo, N.A. 1.4/oil). The
spectrometer slits are configured to operate as an adjustable
longpass filter. At each excitation wavelength, the slits are
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positioned such that the cutoff wavelength is 10 nm longer
than the excitation wavelength. This approach optimizes the
detection sensitivity while blocking elastic scattering at the
excitation wavelength. Figure 8a displays a composite image of
the mixture of beads. The blue, green and red beads were
detected using fluorescence excitation at 508 nm, 560 nm and
632 nm, respectively. This result demonstrates the excellent
contrast that an excitation scan can provide for a mixture of
fluorophores. The measured excitation spectra for the different
beads are plotted in Fig. 8b. Each spectrum is an average of
three beads with the same colour. This ability to multiplex the
detection of multiple fluorophores is a powerful tool that can
provide simultaneous measurements of different phenomena
in biological samples.
Spatial resolution and chromatic aberrations
A broad range of fluorescence excitation and detection
wavelengths is used in this confocal microscope system, and it
is important to consider chromatic aberrations and variations
in spatial resolution with wavelength. To evaluate these issues,
we compare the widths of the point spread functions (PSFs) in
the blue and red spectral regions. The PSFs are determined from
3D confocal images of blue and red fluorescent subresolution
microbeads (0.18-µm diameter) using excitation wavelengths
of 490 nm and 620 nm, respectively. Each 3D image contains
60–100 microbeads and is recorded with the same 60× oil
immersion lens as above and a 150−µm confocal aperture.
The dimensions of the images are 512 × 512 × 64 pixels, and
the projected pixel size is 0.046 µm × 0. 046 µm × 0.050 µm.
The PSF is commonly determined from images of subresolution
microbeads by measuring spatial profiles of the fluorescence
signal from individual microbeads and averaging the profile
widths of multiple beads (Kozubek, 2001). We use a highly
efficient alternative approach to determine the width of the
PSF from multiple bead images. This method consists of two
steps. First, the autocorrelation of the PSF is determined by
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J. H . F R A N K E T A L .
(b)
(a)
z
x
Fig. 9. (a) x–z plane of the autocorrelation of the 3D point spread function. (b) Comparison of autocorrelation along the lateral (x-axis) and axial (z-axis)
directions using fluorescence excitation wavelengths of 490 nm and 620 nm.
computing the 3D spatial autocorrelation of a confocal image
containing multiple microbead images. Second, the width of
the PSF is computed from the autocorrelation of the PSF. The
3D autocorrelation, A(x, y, z), is defined as
A(x, y, z) =
L −1 M−1
N−1
S(i , j , k)S(i − x, j − y, k − z),
Table 1. Point spread function FWHM.
λ excitation (nm)
Lateral
Axial
490
620
0.33 µm
0.36 µm
0.68 µm
0.77 µm
i =0 j =0 k=0
(4)
where S(i , j , k) is the signal at pixel i, j, k in the image of
dimensions L, M, N. For computational efficiency, we perform
the autocorrelation in the frequency domain using
(5)
A(x, y, z) = F F T −1 |F F T (S(i , j , k))|2 ,
where FFT and FFT −1 are the forward and inverse fast
Fourier transforms, respectively. The dominant feature in the
autocorrelation is the central peak, which corresponds to the
self-correlation of the microbead images and is equivalent
to the autocorrelation of the PSF. The correlation between
different bead images produces weak secondary noise peaks
in the autocorrelation. The magnitude of the secondary peaks
is negligible compared to the self-correlation peak because
the beads are randomly distributed in the confocal image and
therefore the spacing between different beads is uncorrelated.
If the confocal image of the beads has a relatively low signal-tonoise ratio (SNR), there is also a noise peak at the centre of the
self-correlation peak that corresponds to the self-correlation of
the noise. The magnitude of this noise peak has been minimized
by increasing the SNR of the confocal image with longer pixel
dwell times and averaging of multiple scans.
Figure 9a shows the x–z plane from a central slice of the
autocorrelation using 490-nm excitation. The autocorrelation
is elongated in the axial direction (z-axis). The autocorrelation
profiles along the lateral and axial directions are shown in
Fig. 9b for the blue and red fluorescent microbeads. The
autocorrelation is inherently broader than the PSF, and the
width of the PSF is estimated from the autocorrelation function
using a scaling relationship from ideal imaging optics. For ideal
diffraction-limited lenses with a circular aperture, the PSF is an
airy disk. The full width at half maximum (FWHM) of the airy
disk is approximately a factor of 0.76 times smaller than the
FWHM of the autocorrelation of the airy disk. We estimate the
width of the PSFs by multiplying the FWHM of the measured
autocorrelations by 0.76. Table 1 shows the PSF widths for the
x and z axes for 490-nm and 620-nm fluorescence excitation.
The axial PSF widths are approximately a factor of two larger
than the lateral PSF widths. In both the axial and lateral
dimensions, the FWHM at 620 nm is 10–15% larger than
at 490 nm. We expect that the measured FWHM values are
a conservative estimate of the actual PSF widths that would
be measured using an ideal point source because the size of
the microbeads is significant compared to the FWHM values
(Kozubek, 2001).
The results of the PSF measurements indicate that
chromatic aberrations are not a significant limitation for
highly resolved multispectral confocal imaging throughout
the visible spectrum and underline the excellent suitability
of PCF-generated supercontinua for confocal microscopy
applications. For further verification, we quantified the
longitudinal chromatic aberrations by measuring the
displacement along the z-axis that was introduced by imaging
at different wavelengths. In these measurements, the reflection
from a glass coverslip was detected as the position of the
coverslip was scanned along the z-axis. At each wavelength,
the optimal focus was defined as the z-position with the
maximum signal from the coverslip. The results showed that
the axial location of the focus varied by +/–0.30 µm for
illumination wavelengths between 470 nm and 650 nm.
C 2007 The Royal Microscopical Society
Journal compilation No claim to original US government works, Journal of Microscopy, 227, 203–215
W H I T E L I G H T C O N F O C A L M I C RO S C O P E
This displacement is comparable to the FWHM of the
PSF in Table 1. We conclude that longitudinal chromatic
aberrations and chromatic variations in beam divergence
are not a significant limitation to this multispectral confocal
microscope system. This result offers exciting opportunities
for applications that require precisely overlapped illumination
at different wavelengths, such as fluorescence colocalization
studies (Manders et al., 1993; Bolte & Cordelieres, 2006),
fluorescence resonance energy transfer (FRET), fluorescence
cross-correlation microscopy and coherent anti-Stokes Raman
scattering (CARS) microscopy. The complete control over laser
intensity and wavelength also makes this instrument ideal for
photoactivation experiments.
1
2
211
4
3
(a)
(b)
0 (a/d counts) 4000
Spectrofluorometric confocal imaging of plants
Highly resolved spectrofluorometric confocal imaging is a
powerful tool for a broad range of microscopy applications.
As an illustration of this capability, we have performed
fluorescence excitation scans with both 2D and 3D confocal
imaging of a thin section of the rhizome of Convallaria Majalis
(lily-of-the-valley) that was stained with safranin and fast
green dyes. A complete 2D fluorescence excitation scan from
480 to 640 nm is available online (see Convallaria movie).
Figures 10a and b show sample 2D fluorescence measurements
of Convallaria at excitation wavelengths of 620 nm and 530 nm,
respectively, corresponding to the peak excitation wavelengths
of fast green and safranin (Li et al., 2002; Angeles et al.,
2004). The fluorescence image at each excitation wavelength
highlights different structures because the fast green and
safranin dyes have affinities for different parts of the sample.
In Fig. 10a, fluorescence signals from fast green are observed
in many different regions of the rhizome. There is a thickened
endodermis, which delimits the wide cortex from the pith. In
the pith, there are scattered vascular bundles that contain
xylem and phloem (Bowes, 1999).
To demonstrate that fluorescence excitation properties
can be used to distinguish different structures, we consider
four subregions of the image in Fig 10a, and show the
corresponding excitation spectra for these structures in Fig
10c. Region 1 is part of the endodermis. Region 2 contains
phloem, and regions 3 and 4 are different regions of xylem.
In the excitation spectra, the narrower peak at 620 nm
corresponds to fast green absorption (Li et al., 2002) and occurs
in all regions. Fast green stains the cellulosic cell walls that
are present in all four regions (Johansen, 1940). The broader
spectral peak that spans 510–530 nm is absorption by safranin
(Angeles et al., 2004), and is only significant for regions 1
and 3. The endodermis and xylem contain lignin, which is
stained by the safranin dye. Note that region 4, which contains
xylem, shows negligible safranin absorption, indicating that
there is differential staining of safranin in different parts of
the xylem, as is evident in Fig. 10b. These spectrally resolved
C 2007 The Royal Microscopical Society
Journal compilation No claim to original US government works, Journal of Microscopy, 227, 203–215
(c)
Fig. 10. (a, b) Two-dimensional confocal fluorescence images of
Convallaria (lily-of-the-valley) rhizome with excitation at 620 nm and
530 nm for peak excitation of fast green and safranin, respectively. Image
is 355 µm × 355 µm. Regions in (a) correspond to (1) endodermis, (2)
phloem and (3, 4) xylem. (c) Fluorescence excitation spectra of the four
regions of interest.
fluorescence measurements enable feature identification by
providing a high degree of labelling specificity.
It is important that the integrated multispectral confocal
microscope system retains the full functionality of the
original confocal microscope. As a demonstration of the
resolution capabilities of our modified CLSM system, highresolution fluorescence and broadband transmission images
of Convallaria are shown in Figs. 11a and b, respectively. These
images resolve the detailed structure of the Convallaria cell
walls, indicating excellent spatial resolution.
As a second demonstration of the CLSM capabilities, we
performed 3D confocal imaging using different fluorescence
excitation wavelengths. Figures 11c–e displays sample 3D
fluorescence images that were measured with excitation
wavelengths of 500 nm, 540 nm and 580 nm, respectively.
The image dimensions are 300 × 200 × 26 voxels with
each voxel corresponding to 0.46 × 0.46 × 0.70 µm3 . These
images show the detailed 3D structure of the Convallaria
with preferential labelling of different structures with different
excitation wavelengths, as described above for the 2D images
in Fig. 10.
212
J. H . F R A N K E T A L .
Fig. 11. High-resolution confocal (a) fluorescence and (b) broadband transmission images of Convallaria rhizome. The colour scale for (a) is the same as
in Figs. 10a, b. (c–e) Comparison of 3D confocal fluorescence measurements of Convallaria with excitation wavelengths at 500, 540 and 580 nm. The
imaged volume is 92 µm × 138 µm × 18 µm.
Spectrofluorometric confocal imaging of live cells
Spectrofluorometric imaging measurements are particularly
powerful when applied to studies of living cells. Live-cell
imaging requires that spectral scans are performed on a
relatively fast timescale. As a demonstration of this capability,
we performed a fluorescence excitation scan of live human
U2OS Osteosarcoma cells that were transiently transfected
with a plasmid expressing an mCherry-Rad51 fusion protein.
Rad51 is a DNA-binding protein that is involved in DNA
repair. The fluorescence excitation scan was measured from
537 to 607 nm using 2D confocal imaging. Figures 12a–
c display sample images of a live cell using excitation
wavelengths of 572, 587 and 602 nm. In the nucleus,
distinctive entangled filament structures are produced by the
overexpressed mCherry-Rad51 fusion protein forming fibrils
around strands of DNA. In contrast, the cytoplasm shows
regions of relatively homogeneous fluorescence signal as well
as regions interspersed with aggregates (Pellegrini et al., 2002).
There are no fibrils in the cytoplasm due to the absence of DNA.
The full fluorescence excitation scan (see live-cell movie) was
performed with 5-nm wavelength increments at a resolution of
512 × 512 pixels with a two-frame Kalman filter at an average
rate of 8.0 s/frame. The time required for confocal scanning
and acquisition of a single frame was 5.4 s. The remaining
2.6 s for each frame was consumed by triggering the confocal
microscope electronics, stepping the excitation wavelength
and measuring the laser power. Further optimization of the
integrated microscope system will enable increased frame
rates.
mCherry is widely accepted as the best available genetically
encoded monomeric fluorescent reporter protein in the red
spectral range (Shaner et al., 2005). The development of
such a monomeric red fluorophore that is suitable for livecell imaging of protein–protein interactions is important for
three main reasons: multiplexing with other green fluorescent
protein (GFP)-tagged proteins in localization studies, providing
a cross-correlation pair for GFP and an acceptor FRET pair for
Venus/yellow fluorescent protein (YFP). Previously, one of the
main problems with mCherry was its relatively poor brightness
as compared to GFP. The brightness, ε, of a fluorescent protein is
defined as the product of its extinction coefficient and quantum
yield. The literature value for the brightness of mCherry is 16
(mMcm)−1 , which is significantly lower than the brightness
of extended yellow fluorescent protein (EYFP) (ε = 51) or
extended green fluorescent protein (EGFP) (ε = 34; Shaner
et al., 2005).
In standard commercial microscopes, the detection
sensitivity for mCherry is further degraded by the lack of
optimal excitation sources. The peak excitation wavelengths
for EGFP and EYFP are approximately 488 nm and 514 nm,
respectively, and optimal excitation is accessible with an Arion laser. Figure 12d shows the measured excitation spectrum
of mCherry within a subregion of the cell in Figs. 12a–c. The
peak excitation wavelength for mCherry is at 590 nm. For the
commercial FluoView system, the nearest laser wavelengths
that are available for excitation are the He-Ne laser at
543 nm and 633 nm and the Kr-ion laser at 568 nm. For some
commercial systems, the Kr-ion laser is not available, and the
nearest wavelengths are the He-Ne laser lines. The spectrum
C 2007 The Royal Microscopical Society
Journal compilation No claim to original US government works, Journal of Microscopy, 227, 203–215
W H I T E L I G H T C O N F O C A L M I C RO S C O P E
572 nm
(a)
587 nm
(b)
213
602 nm
(c)
0 (a/d counts) 3000
(d)
Fig. 12. (a–c) Confocal images from a fluorescence excitation scan of a human U2OS Osteosarcoma cell transfected with an mCherry-Rad51 fluorescent
protein expressing construct. Image dimensions are 59 µm × 59 µm. Excitation wavelengths are indicated in each image. (d) Normalized fluorescence
excitation spectrum of mCherry-Rad51 from a region within the cytoplasm.
in Fig. 12d shows that excitation with these fixed laser
lines provides significantly lower sensitivity than excitation at
590 nm. For example, excitation of mCherry with a He-Ne laser
at 543 nm is 60% less efficient than excitation at 590 nm. The
supercontinuum laser provides access to the peak excitation
wavelength of approximately 590 nm, thus enabling optimum
detection sensitivity.
This ability to perform in situ fluorescence excitation scans
provides important insight into fluorophore properties in
cell environments. For example, protein aggregation and
association of proteins to cellular substructures may result
in subtle spectral shifts that would be impossible to detect
with fixed wavelength systems. The absorption spectrum of
mCherry is known to shift with changing pH (Shu et al., 2006).
Fluorescence excitation scans with the supercontinuum laser
can be used to detect these shifts.
Conclusions
We have demonstrated spectrofluorometric confocal
imaging using a supercontinuum fibre laser and a prism
spectrometer and have fully integrated these systems with a
commercial confocal microscope. With simple adaptations,
the methodology described here permits any existing fixed
wavelength confocal system to be converted into a platform
C 2007 The Royal Microscopical Society
Journal compilation No claim to original US government works, Journal of Microscopy, 227, 203–215
that provides diffraction-limited spectrofluorometric imaging
capabilities. The system is compact, economical and turn-key,
without affecting the inherent functionality of the confocal
microscope. High-resolution fluorescence imaging is feasible
throughout the visible spectrum with relatively minor effects
of chromatic aberration. Different excitation wavelengths
exhibit excellent parfocality without the need for inserting
corrective optics into the beam path after the collimating
optics from the supercontinuum laser source. Corrections
for variations in excitation laser power are performed on a
frame-by-frame basis, which is important for quantitative
measurements over a wide range of wavelengths. Spectral
imaging capabilities are sufficiently fast to track dynamic
phenomena in living cells and can be improved using
parallel detection arrays. The system facilitates multiplexed
measurements of different fluorophores and can improve
contrast between fluorescent labels and autofluorescence.
This new capability may enable the use of autofluorescence
signatures as a diagnostic tool.
Spectrofluorometric imaging can significantly enhance the
sensitivity of fluorescence measurements by enabling optimal
excitation and detection of fluorophores. For example, FRET
measurements could be improved by minimizing spectral
bleed-through and cross-talk (Elangovan et al., 2003). It is
expected that supercontinuum sources will replace traditional
214
J. H . F R A N K E T A L .
fixed-wavelength lasers in many microscopy applications,
and with full spectral capabilities in confocal microscopy,
biophysical research is entering a new era.
Acknowledgments
This work was supported by grants from the Royal Society,
the Engineering and Physical Sciences Research Council
(EPSRC), and the Higher Education Funding Council for
England (HEFCE). J.H. Frank and C.F. Kaminski acknowledge
support by a PLATFORM grant from the EPSRC. J.H. Frank
acknowledges support by the U.S. Department of Energy,
Office of Basic Energy Sciences, Division of Chemical Sciences,
Geosciences, and Biosciences. Sandia National Laboratories is
a multiprogram laboratory operated by Sandia Corporation,
a Lockheed Martin Company, for the U.S. Department of
Energy under contract DE-AC04-94-AL85000. C.F. Kaminski
is grateful to the Leverhulme Trust for personal sponsorship.
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Video Clip S2. Live Cell movie for Figure 12.
This material is available as part of the online article from:
http://www.blackwell-synergy.com/doi/10.1111/j.13652818.2007.01803.x
(This link will take you to the article abstract)
Supplementary Material
The following supplementary material is available for this
article:
Video Clip S1. Convallaria movie for Figure 10;
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